We study a simple bacteriophage system, the progression of promoter usage after infection by bacteriophage T4, as a model for investigating mechanisms of transcriptional regulation. During infection, different classes of T4 promoters (early, middle, late) are recognized as infection proceeds. Middle transcription is performed by the host RNA polymerase, which is modified by ADP-ribosylation and by the binding of the T4 AsiA protein to sigma70, the specificity subunit of RNA polymerase. Middle transcription by modified polymerase also requires the T4 transcriptional activator, MotA, which binds to the -30 region of the promoter. Curiously, unmodified polymerase alone can transcribe from middle promoters, but it is not activated by MotA. By investigating transcription complexes made by unmodified polymerase and T4-modified polymerase, we have found that unmodified polymerase requires only the far downstream sequence elements of a middle promoter (the -10 region). The phage modifications to the polymerase alter this recognition, resulting in a polymerase that now requires the binding of MotA to the - 30 region in order to unwind the DNA and initiate transcription. We have cloned the asiA gene, purified the AsiA protein, and demonstrated that the presence of AsiA is the only modification required for MotA activation. The protein-DNA complex made in the presence of MotA, AsiA and polymerase is more stable to electrophoresis than that made in the absence of any one of these components. These results suggest that the stability of the modified polymerase-promoter complex is mediated through AsiA. We have investigated the importance of different regions of the T4 middle promoter PuvsX, which has MotA boxes at -30, -35, -51, -70, and -87. We constructed mutant PuvsX promoters and assayed the mutants for transcription and binding. Our results indicate that MotA binds independently to the boxes at -30 and -51, and suggests that the box at - 35 is not important for MotA binding. Promoters with changes to the -51 or -35 MotA boxes are still activated by MotA, but the promoter lacking a MotA box at -30 is inactive, indicating that this binding site is critical for transcription.